A method and apparatus for high throughput determination of binding energies between ligands and proteins

Abstract

A method and device for high-throughput determination of binding energy between ligand and protein, the method comprising: a dissociation equilibrium constant calculation step, comprising correlating soluble protein ratio and free ligand concentration under different temperature and different concentration conditions by using a ligand-protein co-precipitation theoretical model equation, to calculate the dissociation equilibrium constant at different temperatures; a binding energy parameter calculation step, comprising calculating the binding energy parameter according to the Gibbs-Helmholtz equation; and an identification step, comprising identifying the interaction type between the ligand and the protein by using the binding energy parameter. The method provided by the application can realize high-throughput determination of various binding energies between ligand and protein in complex biological samples such as cell lysate, living cells, tissues, blood and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of quantitative proteomics, in particular to a method and device for high-throughput determination of binding energy between ligand and protein. BACKGROUND

[0002] Interactions between ligand and protein play important roles in many biological processes such as signal transduction, cell regulation and enzyme catalysis. The mechanism of interaction between ligand and protein is generally simplified as including binding and dissociation of ligand and protein, but in complex biological systems such as cells, tissues and blood, the interaction between ligand and protein can also be accompanied by the formation, dissociation of protein-protein complex and the conformational change of protein. Binding energy such as ΔG, ΔH, ΔS is a key factor to determine the nature of intermolecular reaction, therefore quantitative measurement of binding energy is often used to study the mechanism of interaction between ligand and protein. Various techniques can be used to measure the binding energy between ligand and protein, such as nuclear magnetic resonance, surface plasmon resonance, fluorescence assay, isothermal titration calorimetry, optical tweezers, theoretical calculation, etc. However, these methods are mainly for purified proteins or based on fluorescent markers of proteins under specific conditions, etc., and have limited scope of application and are not suitable for high-throughput studies such as protein-protein complex interactions, or even ligand-protein interaction mechanisms involving protein conformational changes. Therefore, efficient and high-throughput study of ligand-protein interaction mechanism is a great challenge in current pharmacology and drug discovery.

[0003] Cellular thermal shift assay (CETSA) is a method for studying drug targets by biophysical techniques proposed in 2013. The principle is as follows: each protein has its own melting curve. With the increase of temperature, the protein will denature and precipitate. When the protein binds to a small molecule drug, the stability of the complex protein increases, and the melting curve will move to the right. That is, the amount of undegraded protein will increase at the same temperature, and the protein will denature and precipitate after heating. By detecting the thermodynamic stability difference of the protein by different detection methods, it is determined whether it is a drug target according to the change of the thermal melting temperature Tm of the protein with and without drug. In 2014, Savitski et al. first combined CETSA with quantitative mass spectrometry proteomics technology to establish the TPP (Thermal proteome profiling) method. This work uses an empirical formula to fit the relationship between the proportion of soluble proteins and temperature, thereby quantitatively measuring the thermal melting temperature Tm of the protein with and without drug. And using this quantitative method, 51 target points of the broad-spectrum kinase inhibitor staurosporine were identified, and the ferrochelatase (FECH) was identified as the off-target of the kinase inhibitor. On the basis of TPP, ITDR and two-dimensional TPP have also been developed. ITDR refers to the addition of different concentrations of ligands at the same temperature point, and two-dimensional TPP is a combination of ITDR at multiple temperature points.

[0004] The thermal proteome analysis method based on cellular thermal shift assay developed in recent years can be used for high-throughput study of the interaction between ligands and proteins in cell lysates and living cells. However, because there is no specific theoretical model, the parameters of the function do not have a clear physical meaning, so it cannot quantitatively measure the binding energy between ligands and proteins, and cannot accurately identify the ligand-protein interaction containing protein complex formation and dissociation, and even containing protein allosteric. In addition, the existing methods for measuring protein-ligand interaction can usually only reflect the binding strength of the two through the EC50 (half maximal effective concentration) value, but EC50 is not a essential parameter of ligand-protein interaction, and the value will change with the concentration of the protein. Therefore, how to high-throughput quantitatively measure the binding energy in complex biological samples and accurately identify the complex ligand-protein interaction has become a problem to be solved at present. SUMMARY

[0005] According to a first aspect, in an embodiment, a method for high-throughput determining the binding energy between a ligand and a protein is provided, comprising:

[0006] The dissociation equilibrium constant calculation step includes using a ligand-protein co-precipitation theoretical model equation to associate the soluble protein ratio and free ligand concentration under different temperature and different concentration conditions to calculate the dissociation equilibrium constant at different temperatures.

[0007] The binding energy parameter calculation step includes calculating the binding energy parameter according to the Gibbs-Helmholtz equation.

[0008] The recognition step includes recognizing the interaction type between the ligand and the protein according to the binding energy parameter.

[0009] According to a second aspect, in an embodiment, there is provided an apparatus for high-throughput determination of binding energy between a ligand and a protein, comprising:

[0010] The dissociation equilibrium constant calculation module is configured to use a ligand-protein co-precipitation theoretical model equation to associate the soluble protein ratio and free ligand concentration under different temperature and different concentration conditions to calculate the dissociation equilibrium constant at different temperatures.

[0011] The binding energy parameter calculation module is configured to calculate the binding energy parameter according to the Gibbs-Helmholtz equation.

[0012] The recognition module is configured to recognize the interaction type between the ligand and the protein according to the binding energy parameter.

[0013] According to a third aspect, in an embodiment, there is provided an apparatus for high-throughput determination of binding energy between a ligand and a protein, comprising:

[0014] The memory is configured to store a program.

[0015] The processor is configured to implement the method of any one of the first aspect by executing the program stored in the memory.

[0016] According to a fourth aspect, in an embodiment, there is provided a computer readable storage medium, the medium having a program stored thereon, the program being executable by a processor to implement the method of any one of the first aspect.

[0017] According to the above-mentioned embodiment, the method and apparatus for high-throughput determination of binding energy between a ligand and a protein can realize high-throughput determination of various binding energies between ligand-protein in complex biological samples such as cell lysate, living cells, tissues, blood, etc. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A flowchart for high-throughput determination of binding energy between a ligand and a protein in an embodiment;

[0019] Figure 2Fitting graphs of the soluble protein ratio of CDK5, PRKAR2B, STK3 at different temperatures versus the free ligand concentration and fitting graphs of the dissociation equilibrium constant. DETAILED DESCRIPTION

[0020] The application will be further described in details below with specific embodiments and drawings. In the following embodiments, many details are described in order to make the application better understood. However, one skilled in the art can easily recognize that some features can be omitted in different cases, or can be replaced by other materials or methods. In some cases, some operations related to the application are not shown or described in the specification in order to avoid the core part of the application being overwhelmed by too much description, and it is not necessary to describe these related operations in detail for one skilled in the art according to the description in the specification and general technical knowledge in the art.

[0021] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate way to form various embodiments. At the same time, the steps or actions in the method description can also be sequentially adjusted or adjusted in a manner that one skilled in the art can easily see. Therefore, the various sequences in the specification and drawings are only for the purpose of clearly describing a certain embodiment, and do not mean that the sequence is necessary, unless otherwise stated that a certain sequence must be followed.

[0022] The serial numbers of components in this paper, such as "first", "second", etc., are only used to distinguish the described objects, and do not have any sequence or technical meaning.

[0023] As used herein, "room temperature" means 23±2°C.

[0024] According to the first aspect, in an embodiment, a method for determining the binding energy between a ligand and a protein with high throughput is provided, comprising:

[0025] The dissociation equilibrium constant calculation step comprises correlating the soluble protein ratio and the free ligand concentration under different temperature and different concentration conditions using a ligand-protein co-precipitation theoretical model equation, and calculating the dissociation equilibrium constant at different temperatures;

[0026] The binding energy parameter calculation step comprises calculating the binding energy parameter according to the Gibbs-Helmholtz equation (also known as the Gibbs-Helmholtz equation);

[0027] The identification step comprises identifying the interaction type between the ligand and the protein according to the binding energy parameter.

[0028] In one embodiment, the present invention does not require the use of solvents, only through heating, and can quantitatively measure the affinity of drugs to multiple targets at one time. In addition, the existing method measures the EC50 value, which changes with the concentration of the protein. The present invention uses a new formula to fit the dissociation constant K between the ligand and the protein. d This value does not change with the concentration of ligand and protein and is an intrinsic parameter of ligand-protein interaction.

[0029] Existing methods are not suitable for complex biological environments. They are generally one or more purified proteins and require the concentration of the protein to be tested. The method of the present invention is applicable to complex biological environments such as cell lysates, living cells, and tissues, which contain thousands of proteins.

[0030] In one embodiment, in the dissociation equilibrium constant calculation step, the construction of the ligand-protein co-precipitation theoretical model includes: 1) the denaturation precipitation process of the native protein; 2) the process of the ligand and protein forming a ligand-protein complex; 3) the denaturation precipitation process of the ligand-protein complex as an independent entity.

[0031] In one embodiment, in the dissociation equilibrium constant calculation step, the coprecipitation model formula is S=S0+(S ∞ -S0)×a×[L] / (1+a×[L]), K d =S0 / (a*S ∞ ); S is the ratio of soluble protein, S0 is the ratio of soluble protein at the test temperature without adding ligand, which can be measured experimentally; S ∞ K is the ratio of soluble protein when the ligand concentration is infinite at the test temperature, which is obtained by fitting the coprecipitation model formula; d is the dissociation constant; [L] is the free ligand concentration; a is the fitting constant. The present invention proposes the coprecipitation model formula for the first time, which realizes the calculation of the soluble protein ratio and thus the calculation of the dissociation equilibrium constant. In addition, the dissociation constant K between the ligand and the protein is d And by K d The calculated binding energy does not change with the concentration of the protein and is an essential parameter of the ligand-protein interaction.

[0032] In one embodiment, in the dissociation equilibrium constant calculation step, the soluble protein ratio and free ligand concentration are detected by two-dimensional cell thermal shift analysis.

[0033] In one embodiment, in the dissociation equilibrium constant calculation step, the two-dimensional cell thermal shift analysis method includes performing ligand-drug binding and heating experiments at different temperatures and different ligand concentrations.

[0034] In an embodiment, in the dissociation equilibrium constant calculation step, the temperature is at least 3 of 37-67℃, and the ligand concentration is at least 8 of 0-100 μM.

[0035] In an embodiment, in the dissociation equilibrium constant calculation step, when the soluble protein ratio is detected by the two-dimensional cell thermal shift assay method, the relative abundance of the protein under different temperature and concentration conditions and the relative abundance of the protein under the control temperature are obtained by quantitative mass spectrometry, the relative abundance of the protein under different temperature and concentration conditions is divided by the relative abundance of the protein under the control temperature, and the soluble protein ratio is obtained. In an embodiment, the control temperature can be 37℃.

[0036] In an embodiment, the quantitative mass spectrometry includes adding an isotopic label in different protein samples.

[0037] In an embodiment, the isotopic label includes at least one of the same amount and different sequence label, relative and absolute quantitative isobaric tag for relative and absolute quantification (iTRAQ), dimethylated label.

[0038] In an embodiment, the free ligand concentration refers to the concentration of the ligand that is not combined with the protein in the solution after the ligand is combined with the protein, which is equal to the initial concentration of the ligand minus the concentration of the ligand combined with the protein.

[0039] In an embodiment, the detection method of the free ligand concentration includes but is not limited to at least one of the internal standard method and the external standard method, and the concentration of the uncombined ligand (i.e. the free ligand) in the solution can be measured using the foregoing method. The peak area of the ligand is measured by using one or both of high-resolution mass spectrometry and liquid chromatography, and a standard curve is drawn by using the peak area and the concentration.

[0040] In an embodiment, in the dissociation equilibrium constant calculation step, when the soluble protein ratio and the free ligand concentration are associated by using the theoretical model equation of ligand-protein co-precipitation, the soluble protein ratio and the free ligand concentration are associated by using the co-precipitation model formula, and the dissociation constant K d According to the Gibbs-Helmholtz equation (also known as the Gibbs-Helmholtz equation), the binding energy parameter is calculated.

[0041] In an embodiment, the binding energy parameter includes but is not limited to at least one of enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy (ΔG), etc.

[0042] In one embodiment, in the identifying step, the type of interaction between the ligand and the protein includes, but is not limited to, at least one of the following reactions: (1) classical ligand-protein interaction: i.e., the protein and the ligand only have binding and dissociation reactions; (2) ligand-protein interaction accompanied by protein complex formation: i.e., the target protein binds to another protein in the process of binding to the ligand; (3) ligand-protein interaction accompanied by protein complex dissociation: i.e., the target protein initially exists in the form of a complex, and the protein complex dissociates in the process of binding to the ligand; (4) ligand-protein interaction accompanied by protein conformational change: i.e., the structure of the target protein changes in the process of binding to the ligand.

[0043] In one embodiment, in the identifying step, at least one of the following criteria is used to identify the type of interaction between the ligand and the protein: (1) classical ligand-protein interaction: the enthalpy change is less than 0 kcal-mol -1 and the Gibbs free energy change is between -15 kcal-mol -1 and -7 kcal-mol -1 (inclusive of the end points -15 kcal-mol -1 , -7 kcal-mol -1 ); (2) ligand-protein interaction accompanied by protein complex formation: the enthalpy change is less than 0 kcal-mol -1 and the Gibbs free energy change is less than -20 kcal-mol -1 ; (3) ligand-protein interaction accompanied by protein complex dissociation: the enthalpy change is greater than 0 kcal-mol -1 ; (4) ligand-protein interaction accompanied by protein conformational change: the enthalpy change is less than 0 kcal-mol -1 and the Gibbs free energy change is less than -20 kcal-mol -1 , or the enthalpy change is greater than 0 kcal-mol -1 and the Gibbs free energy change is greater than 0 kcal-mol -1 .

[0044] In one embodiment, in the identifying step, the interaction between the ligand and the protein includes, but is not limited to, the protein-ligand interaction of a cell lysate, a cell, a tissue, or a blood sample.

[0045] In one embodiment, in the identifying step, different types of target points or conformational inhibitors are identified according to the binding energy parameters.

[0046] In one embodiment, in the identifying step, the identifying of the allosteric inhibitor specifically refers to different ligands having different effects on the structure of the same protein target, and the allosteric inhibitor refers to a ligand that has a great change on the structure of the protein, and the identification criterion is consistent with the identification criterion of the ligand-protein interaction accompanying the allosteric change of the protein.

[0047] According to the second aspect, in one embodiment, there is provided a device for high-throughput determination of the binding energy between a ligand and a protein, comprising:

[0048] a dissociation equilibrium constant calculation module configured to calculate the dissociation equilibrium constant at different temperatures by correlating the soluble protein ratio and the free ligand concentration at different temperatures and different concentrations using a theoretical model equation of ligand-protein co-precipitation;

[0049] a binding energy parameter calculation module configured to calculate the binding energy parameter according to the Gibbs-Helmholtz equation;

[0050] an identification module configured to identify the interaction type between the ligand and the protein according to the binding energy parameter.

[0051] According to the third aspect, in one embodiment, there is provided a device for high-throughput determination of the binding energy between a ligand and a protein, comprising:

[0052] a memory configured to store a program;

[0053] a processor configured to implement the method of any one of the first aspect by executing the program stored in the memory.

[0054] According to the fourth aspect, in one embodiment, there is provided a computer readable storage medium, wherein the medium stores a program capable of being executed by a processor to implement the method of any one of the first aspect.

[0055] In one embodiment, there is provided a method for high-throughput determination of the binding energy between a ligand and a protein, which utilizes a theoretical model equation of ligand-protein co-precipitation to correlate the soluble protein ratio and the free ligand concentration measured by two-dimensional cell thermal shift assay technology at different temperatures and different concentrations, to obtain the dissociation equilibrium constant at different temperatures, and then calculates the binding energy parameter according to the Gibbs-Helmholtz equation, and finally identifies the interaction type between the ligand and the protein using the obtained binding energy.

[0056] In one embodiment, the construction of the theoretical model of ligand-protein co-precipitation includes three processes, namely, the denaturation and precipitation process of the native protein, the process of forming a ligand-protein complex by the ligand and the protein, and the denaturation and precipitation process of the ligand-protein complex as an independent whole. The co-precipitation model formula is S=S0+(S ∞-S0) x a x [L] / (1 + a x [L]), wherein K d = S0 / (a * S ∞ ); S0 is the soluble protein ratio without ligand at the temperature to be measured, which can be measured by experiment; S ∞ is the soluble protein ratio when the ligand concentration is infinite at the temperature to be measured, which is obtained by fitting the co-precipitation model formula; K d is the dissociation constant; [L] is the free ligand concentration; and a is a fitting constant.

[0057] In an embodiment, the two-dimensional cell thermal shift assay technology includes ligand and drug binding and heating experiments at different temperatures and different ligand concentrations. The temperature is three or more of 37-67℃, and the ligand concentration is eight or more of 0-100 μM.

[0058] In an embodiment, when measuring the soluble protein ratio by the two-dimensional cell thermal shift assay technology, the relative abundance of proteins under different temperatures and different concentrations is obtained by high-resolution quantitative mass spectrometry technology, and the relative abundance of proteins under a control temperature (for example, 37℃) is obtained. The relative abundance of proteins under different temperatures and different concentrations is divided by the relative abundance of proteins under the control temperature to obtain the soluble protein ratio.

[0059] In an embodiment, the high-resolution quantitative mass spectrometry technology includes one or more of the same mass different sequence labeling technology TMT10plex, TMT11plex, TMT16plex, the relative and absolute quantitative isobaric labeling technology (iTRAQ), and the dimethylation labeling technology.

[0060] In an embodiment, the free ligand concentration refers to the concentration of ligand that is not combined with protein in the solution after the ligand is combined with the protein, which is equal to the initial concentration of the ligand minus the concentration of the ligand combined with the protein.

[0061] In an embodiment, for the detection of the free ligand concentration, one or both of the internal standard method and the external standard method can be used. The concentration of the ligand that is not combined with the protein in the solution (i.e., the free ligand) is detected. The peak area of the ligand is measured by one or both of high-resolution mass spectrometry and liquid chromatography, and a standard curve is drawn using the peak area and the concentration.

[0062] In an embodiment, when correlating the soluble protein ratio and the free ligand concentration by using the ligand-protein co-precipitation theoretical model equation, the dissociation constant K d under different temperatures is obtained by correlating the soluble protein ratio and the free ligand concentration by using the co-precipitation model formula, and then the enthalpy change (ΔH), the entropy change (ΔS), the Gibbs free energy (ΔG), and other energy parameters are obtained according to the Gibbs-Helmholtz equation.

[0063] In one embodiment, the types of ligand-protein interactions include the following: (1) classical ligand-protein interaction: only binding and dissociation reactions exist between the protein and the ligand; (2) ligand-protein interaction with protein complex formation: the target protein binds to another protein during the binding of the ligand to the target protein; (3) ligand-protein interaction with protein complex dissociation: the target protein initially exists in the form of a complex, and the protein complex dissociates during the binding of the ligand to the target protein; (4) ligand-protein interaction with protein conformational change: the structure of the target protein changes greatly during the binding of the ligand to the target protein; and (5) one or more of the above combinations.

[0064] In one embodiment, the types of ligand-protein interactions are identified based on the following criteria: (1) classical ligand-protein interaction: the enthalpy change is less than 0 kcal-mol -1 , and the Gibbs free energy change is between -15 kcal-mol -1 and -7 kcal-mol -1 ; (2) ligand-protein interaction with protein complex formation: the enthalpy change is less than 0 kcal-mol -1 , and the Gibbs free energy change is less than -20 kcal-mol -1 ; (3) ligand-protein interaction with protein complex dissociation: the enthalpy change is greater than 0 kcal-mol -1 ; (4) ligand-protein interaction with protein conformational change: the Gibbs free energy change is less than -20 kcal-mol -1 or the enthalpy change is greater than 0 kcal-mol -1 .

[0065] In one embodiment, the method for measuring the binding energy between ligands and proteins provided by the present application can be applied to the analysis of protein-ligand interactions in cell lysates, cells, tissues, or blood samples.

[0066] In one embodiment, the method for measuring the binding energy between ligands and proteins can be used to measure the dissociation constant and binding energy of ligands and targets in complex biological samples, identify different types of targets, and identify allosteric inhibitors.

[0067] In one embodiment, the identification of allosteric inhibitors specifically refers to different ligands having different effects on the structure of the same protein target. An allosteric inhibitor refers to a ligand that greatly changes the structure of the protein, and the identification criteria are consistent with those for ligand-protein interactions with protein conformational changes.

[0068] In an embodiment, a method for high-throughput determination of binding energy between ligand-protein is provided, which combines two-dimensional cell thermal shift assay technology and a co-precipitation model, and can be used for high-throughput quantitative measurement of binding energy between proteins and ligands in complex biological environments (cell lysate, living cells, etc.).

[0069] In an embodiment, the present application provides a method for high-throughput determination of binding energy between ligand-protein, which uses a theoretical model equation of ligand-protein co-precipitation to correlate the soluble protein ratio and free ligand concentration measured by two-dimensional cell thermal shift assay at different temperatures and different concentrations, obtains dissociation equilibrium constants at different temperatures, calculates the binding energy according to Gibbs Helmholtz equation, and finally uses the obtained binding energy to explain the interaction mechanism between ligand-protein. The specific process is as follows: (1) measuring the concentration of free ligand after ligand-protein binding by high-resolution mass spectrometry; (2) measuring the soluble ratio of each protein at different temperatures and different concentrations by high-resolution quantitative mass spectrometry; (3) establishing a theoretical model of ligand-protein co-precipitation; (4) using the theoretical model of co-precipitation to correlate the free ligand concentration and the soluble protein ratio to obtain energy-related parameters, including dissociation constant (K d ), Gibbs free energy (ΔG), enthalpy change (ΔH), entropy change (ΔS), etc.; (5) identifying the type of ligand-protein interaction according to the binding energy, including the binding and dissociation of protein complex, and the allosteric effect of protein. The method provided by the present application can realize high-throughput determination of various binding energies between ligand-protein in complex biological samples, including cell lysate, living cells, tissues, blood, etc.

[0070] In an embodiment, the present application provides a method for high-throughput determination of binding energy between ligand-protein. By using the formula obtained by the co-precipitation model provided by the present application, combined with two-dimensional cell thermal shift assay technology, the dissociation constant (K d ), Gibbs free energy (ΔG), enthalpy change (ΔH), entropy change (ΔS) of ligand and protein in complex biological samples can be measured, and high-throughput and accurate quantitative measurement of ligand-protein interaction can be realized. In addition, the method for determination of binding energy between ligand-protein provided by the present application can accurately identify different types of targets and allosteric inhibitors in complex biological samples.

[0071] In an embodiment, the present application adopts the following technical solutions:

[0072] (1) Two-dimensional cell thermal shift assay: A series of ligand drugs with different concentrations (0-100 mM, the number of concentrations is N) are incubated with cell lysate, tissue lysate, etc. at room temperature to complete the drug administration experiment. Then heat at M different temperatures (46°C, 48°C, 50°C, 52°C, 54°C) to make the proteins in the lysate precipitate. Set the control group at 37°C at each temperature, and the drug concentration is 0 mM. A total of M x (N+1) samples are obtained at M different temperatures, and batch high-speed centrifugation is performed to obtain the supernatant. Divide the supernatant of each sample into two parts.

[0073] (2) Protein sample pretreatment experiment and mass spectrometry analysis: Take 10-30 mg of the first M x (N+1) sample supernatant for solution digestion to obtain a peptide mixture. Then, the peptide mixture is labeled by quantitative mass spectrometry tag. The labeled samples at each temperature are combined and fractionated. Finally, high-resolution mass spectrometry is collected, and the solubility ratio of each protein at different concentrations and different temperatures is obtained by quantitative proteomics technology.

[0074] (3) Ligand purification and free ligand concentration determination: The second M x (N+1) sample supernatant is adsorbed on a C18 Tip column to make the ligand drugs adsorbed on the C18 Tip column by hydrophobic interaction. After washing the impurities, the ligand drugs adsorbed on the C18 Tip column are eluted to obtain M x (N+1) eluate. After freeze-drying, high-resolution mass spectrometry is collected, the peak area is calculated, and the free ligand concentration at different temperatures and different concentrations is obtained.

[0075] (4) Correlate the solubility of proteins with the free ligand concentration using the co-precipitation model to obtain energy parameters: Substitute the solubility of proteins and the free ligand concentration into the co-precipitation model S = S0 + (S ∞ -S0) x a x [L] / (1 + a x [L]), where K d =S0 / (a * S ∞ ), to obtain the dissociation constant K d , and substitute it into the Gibbs-Helmholtz equation to calculate the binding energy ΔG, ΔH, ΔS, etc.

[0076] Example 1

[0077] Figure 1 The flowchart of high-throughput determination of ligand-protein binding energy in this embodiment is shown.

[0078] The specific steps are as follows:

[0079] (1) Two-dimensional cell thermal shift experiment: A series of different concentrations (100 μM, 25 μM, 6.25 μM, 1.5625 μM, 0.3906 μM, 0.0976 μM, 0.0244 μM, 0.006 μM, 0.0015 μM, 0 μM) of a broad-spectrum protein kinase inhibitor (Staurosporine) were incubated with K562 cell lysate at room temperature to complete the drug administration experiment. Subsequently, the cells were heated at 5 different temperatures (46°C, 48°C, 50°C, 52°C, 54°C) to precipitate the proteins in the lysate. A control group was set at 37°C at each temperature, and the drug concentration was 0 μM. A total of 55 samples were obtained at 5 different temperatures, and the samples were centrifuged at high speed in batches to obtain the supernatant. The supernatant of each sample was divided into two parts. The first supernatant was used for proteomic sample pretreatment experiments, and the second supernatant was used for free ligand purification.

[0080] (2) Proteomic sample pretreatment experiment and mass spectrometry analysis: 20 μg of the supernatant of the first 55 samples was taken out for solution enzymatic hydrolysis to obtain a peptide mixture. Then, the peptide mixture was labeled with TMT11plex. After desalting, the eluates of 11 samples at each temperature were combined to obtain a total of 5 combined eluates. After freeze-drying, the 5 eluates were sequentially subjected to a C18 Tip column-based fractionation experiment, and each eluate was divided into 6 fractions, for a total of 30 fractions. Finally, the 30 fractions were sequentially subjected to high-resolution mass spectrometry acquisition, and the solubility ratio of each protein at different concentrations and temperatures was obtained by quantitative proteomics technology.

[0081] (3) Ligand purification and free ligand concentration determination: The supernatants of the second batch of 55 samples were adsorbed on C18 Tip cartridges, so that the broad-spectrum protein kinase inhibitor Staurosporine was adsorbed on the C18 Tip cartridges through hydrophobic interaction. After cleaning the impurities, the broad-spectrum protein kinase inhibitor Staurosporine adsorbed on the C18 Tip cartridges was eluted to obtain 55 eluates. After freeze-drying, an equal amount of internal standard with a similar structure to Staurosporine was added to the 55 samples, and then high-resolution mass spectrometry was performed. A standard curve of Staurosporine was drawn, and the free ligand concentration at different temperatures and concentrations was calculated based on the peak area ratio of the ligand to the internal standard.

[0082] (4) Use the coprecipitation model to correlate the soluble protein ratio and the free ligand concentration to obtain the energy parameter: Substitute the soluble protein ratio and the free ligand concentration into the coprecipitation model S = S0 + (S ∞ -S0)×a×[L] / (1+a×[L]), where K d =S0 / (a*S ∞), to obtain dissociation constant K d and substituting it into the Gibbs Helmholtz equation to calculate the binding energy ΔG, ΔH, ΔS and other parameters. Through the method of the present application, more than 60 kinase target proteins are identified at 46°C, 48°C, 50°C, 52°C and 54°C, and the energy parameters of each kinase target protein can be obtained. Taking CDK5, PRKAR2B and STK3 proteins as examples, their soluble protein ratio and free ligand concentration fitting curve and dissociation equilibrium constant fitting are shown in Figure 2

[0083] Example 2

[0084] (1) Two-dimensional cell thermal shift experiment: a series of different concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.7812 μM, 0.3906 μM, 0.1953 μM, 0.0976 μM, 0.0488 μM, 0.0244 μM, 0.0122 μM, 0 μM) of AKT protein orthosteric inhibitor (AT7867) were incubated with HKE 293T cell lysate at room temperature to complete the drug administration experiment. Then heating was carried out at 4 different temperatures (48°C, 50°C, 52°C, 54°C) to precipitate the proteins in the lysate. The control group was set at 37°C at each temperature, and the drug concentration was 0 μM. A total of 64 samples were obtained at 4 different temperatures, and high-speed centrifugation was carried out in batches to obtain the supernatant. The supernatant of each sample was divided into two parts, the first part of the supernatant was used for proteomic sample pretreatment experiment, and the second part of the supernatant was used for free ligand purification.

[0085] (2) Proteomic sample pretreatment experiment and mass spectrometry analysis: 20 μg of the first 64 sample supernatants were taken out for solution digestion to obtain peptide mixtures. Then, the peptide mixtures were labeled with TMT16plex. After desalination, the eluents of 16 samples at each temperature were combined to obtain 4 combined eluents. After freeze-drying, the 4 eluents were sequentially subjected to C18 Tip column-based fractionation experiment, and each eluent was divided into 8 fractions, a total of 32 fractions. Finally, the 32 fractions were sequentially subjected to high-resolution mass spectrometry acquisition, and the solubility ratio of each protein at different concentrations and different temperatures was obtained by quantitative proteomics technology.

[0086] ​(3) Ligand purification and free ligand concentration determination: The second 64 samples of supernatant were adsorbed by C18 Tip column respectively, so that AKT protein orthosteric inhibitor (AT7867) was adsorbed on the C18 Tip column by hydrophobic interaction. After washing the impurities, the AKT protein orthosteric inhibitor (AT7867) adsorbed on the C18 Tip column was eluted to obtain 64 eluents. After freeze-drying, an equal amount of internal standard similar in structure to AT7867 was added to the 64 samples, followed by high-resolution mass spectrometry acquisition. Draw the AT7867 standard curve, and calculate the free ligand concentration at different temperatures and different concentrations by the peak area ratio of ligand to internal standard.

[0087] (4) Correlate the proportion of soluble protein with the free ligand concentration using the co-precipitation model to obtain energy parameters: Substitute the proportion of soluble protein and the free ligand concentration into the co-precipitation model S = S0 + (S ∞ -S0) × a × [L] / (1 + a × [L]), K d = S0 / (a * S ∞ ), to obtain the dissociation constant K d , and substitute it into the Gibbs Helmholtz equation to calculate the binding energy ΔG, ΔH, ΔS, etc. parameters. Taking AKT1 protein as an example, the energy parameters obtained are shown in Table 1. The energy parameters indicate that the interaction between AT7867 and AKT1 is non-allosteric, i.e. a classic ligand-protein interaction, also known as a classic non-allosteric protein interaction.

[0088] Table 1 Binding energy parameters of AKT1 and AT7867

[0089] Protein ΔH ΔS ΔG(298.15 K) Type of interaction AKT1 -21.345 kcal-mol -1 ]] -0.037 kcal-mol -1 ]] -10.416 kcal-mol -1 ]] Non-allosteric

[0090] Example 3

[0091] (1) Two-dimensional cell thermal shift experiment: A series of different concentrations (100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, 0.00128 μM, 0 μM) of AKT protein allosteric inhibitor (MK-2206) were incubated with HeLa cell lysate at room temperature to complete the drug administration experiment. Then heating was carried out at 5 different temperatures (46°C, 48°C, 50°C, 52°C, 54°C) to make the proteins in the lysate precipitate. The control group was set at 37°C at each temperature, and the drug concentration was 0 μM. A total of 50 samples were obtained at 5 different temperatures, and high-speed centrifugation was carried out in batches to obtain the supernatant. The supernatant of each sample was divided into two parts, the first part of the supernatant was used for protein group sample pretreatment experiment, and the second part of the supernatant was used for free ligand purification.

[0092] (2) Protein sample pretreatment experiment and mass spectrometry analysis: 20 μg of supernatant of the first 50 samples was taken out for solution digestion to obtain a peptide mixture. Then, the peptide mixture was labeled with TMT10plex. After desalination, the eluate of 10 samples at each temperature was combined to obtain 5 combined eluates. After freeze-drying, the 5 eluates were sequentially subjected to C18 Tip column-based fractionation experiment, and each eluate was divided into 6 fractions, a total of 30 fractions. Finally, the 30 fractions were sequentially subjected to high-resolution mass spectrometry acquisition, and the solubility ratio of each protein at different concentrations and different temperatures was obtained by quantitative proteomics technology.

[0093] (3) Ligand purification and free ligand concentration determination: the supernatant of the second 50 samples was adsorbed on a C18 Tip column to make the AKT protein allosteric inhibitor (MK-2206) adsorbed on the C18 Tip column by hydrophobic interaction. After removing impurities, the AKT protein allosteric inhibitor (MK-2206) adsorbed on the C18 Tip column was eluted to obtain 50 eluates. After freeze-drying, an equal amount of an internal standard similar in structure to MK-2206 was added to the 50 samples, followed by high-resolution mass spectrometry acquisition. A standard curve of MK-2206 was drawn, and the free ligand concentration at different temperatures and different concentrations was calculated by the peak area ratio of the ligand to the internal standard.

[0094] (4) Correlate the solubility protein ratio with the free ligand concentration using the co-precipitation model to obtain energy parameters: the solubility protein ratio and the free ligand concentration are substituted into the co-precipitation model S = S0 + (S ∞ -S0) × a × [L] / (1 + a × [L]), where K d = S0 / a / S ∞ , to obtain the dissociation constant K d , and substitute it into the Gibbs Helmholtz equation to calculate the binding energy ΔG, ΔH, ΔS, etc. parameters. By the method of the application, more than 60 kinase target proteins are identified at 46°C, 48°C, 50°C, 52°C, and 54°C, and the energy parameters of each kinase target protein can be obtained. Taking AKT1 protein as an example, the energy parameters obtained are shown in Table 2. The energy parameters indicate that the interaction between MK-2206 and AKT1 is allosteric interaction, i.e. the interaction between the ligand and the protein accompanying the formation of a protein complex.

[0095] Table 2 Binding energy parameters of AKT1 and MK-2206

[0096] Protein ΔH ΔS ΔG(298.15 K) Type of interaction AKT1 -389.788 kcal-mol -1 ]] -1.163 kcal-mol -1 ]] -43.034 kcal-mol -1 ]] Allosteric

[0097] Those skilled in the art can understand that all or part of the functions of various methods in the above embodiments can be realized by hardware or by a computer program. When all or part of the functions in the above embodiments are realized by a computer program, the program can be stored in a computer readable storage medium, which can include a read-only memory, a random access memory, a magnetic disk, an optical disk, a hard disk, and the like. The above functions are realized by executing the program by a computer. For example, the program is stored in a memory of a device, and the above functions are realized by executing the program in the memory by a processor. In addition, when all or part of the functions in the above embodiments are realized by a computer program, the program can also be stored in a storage medium such as a server, another computer, a disk, an optical disk, a flash disk, or a mobile hard disk, and is saved in a memory of a local device by downloading or copying, or the system of the local device is updated, and the above functions are realized by executing the program in the memory by a processor.

[0098] The above application of specific examples to the present application is described, which is only used to help understand the present application and does not limit the present application. For those skilled in the art, according to the idea of the present application, a number of simple deductions, deformations or substitutions can be made.

Claims

1. A device for high-throughput determination of binding energy between a ligand and a protein, characterized in that: include: Dissociation equilibrium constant calculation module for using the ligand-protein coprecipitation theoretical model equation S = S0 + (S ∞ -S0)×a×[ L ] / (1+a×[ L ]) Correlate the soluble protein ratio and free ligand concentration under different temperature and concentration conditions, and then use K d =S0 / (a×S ∞ ) to calculate the dissociation equilibrium constant at different temperatures; where S is the proportion of soluble protein, S0 is the proportion of soluble protein without ligand at the test temperature; S ∞ K is the ratio of soluble protein when the ligand concentration is infinite at the test temperature, which is obtained by fitting the coprecipitation model formula; d is the dissociation equilibrium constant; [ L ] is the free ligand concentration; a is the fitting constant; a binding energy parameter calculation module, configured to calculate a binding energy parameter according to the Gibbs-Helmholtz equation, wherein the binding energy parameter includes at least one of enthalpy change, entropy change, and Gibbs free energy; An identification module is used to identify the type of interaction between the ligand and the protein based on the binding energy parameter; wherein the type of interaction between the ligand and the protein includes at least one of the following reactions: (1) classical ligand-protein interaction: that is, there is only binding and dissociation reaction between the protein and the ligand; (2) ligand-protein interaction accompanied by protein complex formation: that is, during the process of the ligand binding to the target protein, the target protein undergoes a binding reaction with another protein; (3) ligand-protein interaction accompanied by protein complex dissociation: that is, the target protein initially exists in the form of a complex, and during the process of the ligand binding to the target protein, the protein complex undergoes a dissociation reaction; (4) ligand-protein interaction accompanied by protein conformational change: that is, during the process of the ligand binding to the target protein, the structure of the target protein changes.

2. A device for high-throughput determination of binding energy between ligands and proteins, characterized in that: include: Memory, used to store programs; A processor, configured to implement a method for high-throughput determination of binding energy between a ligand and a protein by executing a program stored in the memory, wherein the method for high-throughput determination of binding energy between a ligand and a protein comprises: The dissociation equilibrium constant calculation step includes using the ligand-protein coprecipitation theoretical model equation S = S0 + (S ∞ -S0)×a×[ L ] / (1+a×[ L ]) Correlate the soluble protein ratio and free ligand concentration under different temperature and concentration conditions, and then use K d =S0 / (a×S ∞ ) to calculate the dissociation equilibrium constant at different temperatures; where S is the proportion of soluble protein, S0 is the proportion of soluble protein without ligand at the test temperature; S ∞ K is the ratio of soluble protein when the ligand concentration is infinite at the test temperature, which is obtained by fitting the coprecipitation model formula; d is the dissociation equilibrium constant; [ L ] is the free ligand concentration; a is the fitting constant; The step of calculating the binding energy parameter comprises calculating the binding energy parameter according to the Gibbs-Helmholtz equation, wherein the binding energy parameter comprises at least one of enthalpy change, entropy change, and Gibbs free energy; The identification step includes identifying the type of interaction between the ligand and the protein based on the binding energy parameter; wherein the type of interaction between the ligand and the protein includes at least one of the following reactions: (1) classical ligand-protein interaction: that is, there is only binding and dissociation reaction between the protein and the ligand; (2) ligand-protein interaction accompanied by protein complex formation: that is, during the process of the ligand binding to the target protein, the target protein undergoes a binding reaction with another protein; (3) ligand-protein interaction accompanied by protein complex dissociation: that is, the target protein initially exists in the form of a complex, and during the process of the ligand binding to the target protein, the protein complex undergoes a dissociation reaction; (4) ligand-protein interaction accompanied by protein conformational change: that is, during the process of the ligand binding to the target protein, the structure of the target protein changes.

3. The device according to claim 2, wherein In the dissociation equilibrium constant calculation step, the construction of the ligand-protein co-precipitation theoretical model includes: 1) the denaturation precipitation process of the native protein; 2) the process of the ligand and protein forming a ligand-protein complex; and 3) the denaturation precipitation process of the ligand-protein complex as an independent entity.

4. The device according to claim 2, wherein In the dissociation equilibrium constant calculation step, the soluble protein ratio and free ligand concentration are detected by a two-dimensional cell thermal shift analysis method.

5. The device according to claim 4, characterized in that In the dissociation equilibrium constant calculation step, the two-dimensional cell thermal shift analysis method includes performing ligand-drug binding and heating experiments at different temperatures and different ligand concentrations.

6. The device according to claim 5, characterized in that In the dissociation equilibrium constant calculation step, the temperature is at least three of 37 to 67° C., and the ligand concentration is at least eight of 0 to 100 μM.

7. The device according to claim 4, wherein In the dissociation equilibrium constant calculation step, when detecting the soluble protein ratio by a two-dimensional cell thermal shift analysis method, the relative abundance of proteins under different temperature and different concentration conditions and the relative abundance of proteins at a control temperature are obtained by quantitative mass spectrometry, and the relative abundance of proteins under the different temperature and different concentration conditions is divided by the relative abundance of proteins at the control temperature to obtain the soluble protein ratio.

8. The device according to claim 7, wherein The quantitative mass spectrometry involves adding isotopic tags to different protein samples.

9. The device according to claim 8, wherein The isotopic tag includes at least one of an isobaric tag, an isobaric tag for relative and absolute quantification, and a dimethylated tag.

10. The device according to any one of claims 2 to 9, characterized in that: The free ligand concentration refers to the concentration of the ligand not bound to the protein in the solution after the ligand binds to the protein, which is equal to the initial concentration of the ligand minus the concentration of the ligand bound to the protein.

11. The device according to any one of claims 2 to 9, characterized in that: In the identification step, when identifying the type of interaction between the ligand and the protein, at least one of the following identification criteria is used: (1) Classical ligand-protein interaction: enthalpy change less than 0 kcal·mol -1 , and the Gibbs free energy change is -15 kcal·mol -1 to -7 kcal·mol -1 (2) Ligand-protein interaction accompanying protein complex formation: enthalpy change is less than 0 kcal·mol -1 , and the Gibbs free energy change is less than -20 kcal·mol -1 (3) Ligand-protein interaction accompanying protein complex dissociation: enthalpy change greater than 0 kcal·mol -1 ; (4) Ligand-protein interaction accompanied by protein conformational change: enthalpy change is less than 0 kcal·mol -1 , and the Gibbs free energy change is less than -20 kcal·mol -1 , or the enthalpy change is greater than 0 kcal·mol -1 , and the Gibbs free energy change is greater than 0 kcal·mol -1 .

12. The device according to any one of claims 2 to 9, characterized in that In the identification step, the interaction between the ligand and the protein includes the interaction between the protein in the cell lysate, cell, tissue or blood sample and the ligand.

13. The device according to claim 12, wherein The identification step includes identifying different types of targets or allosteric inhibitors based on the binding energy parameters.

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